- ...EpsteinBarr virus DNA relea...
- Foosung(093370)股票行情走势技术分析_未来预测...
- 猪抗凝血酶受体(ATR)elisa试剂盒_
- [PSP]为了守护人类与妖作战!冒险RPG《TSUKUMON...
- 【求助】做荧光定量PCR提RNA用试剂盒还是普通方法好 核...
- Chromosomal Circularization in...
- 细胞计数仪器 | 细胞成像仪 | ViCELL XR 细胞计...
- 手腕带 JJC
- 酵母双杂交系统研究及其应用_liangpanpan1983的...
- 超氧化物歧化酶研究的历史,现状及应用前景
- 国精化学股份有限公司 | pci
- 纯化DNA实验_基因组DNA的快速纯化
- [07-28]求助动物组织的DNA提取试剂盒 00问答网
- [07-23]基因组DNA提取试剂盒| Genomic DNA Isolation Kit细胞分析试剂盒艾...
- [10-03]什么是TENP溶液,它的作用是什么?它是用在微生物DNA的提取过程...
- [10-03]细菌DNA提取方法
- [10-03]DNA提取液的配制
- [07-24]动物组织DNA提取方法
- [10-02]DNA分离纯化学术百科知网空间
- [07-30]国内能买到的快速高效提取人DNA试剂盒有哪些?
- [10-03]常规DNA纯化试剂盒(PCR产物,酶切产物)50T_价格厂家供应商_...
Preparation of Electrocompetent Cells and Electroporation of Plasmid DNA
PreparationofElectrocompetentCellsandElectroporationofPlasmidDNA
- Inoculate5-mlL-brothwithasinglecolonyofE.coli.Incubate5hourstoovernightat37°Conarollerorwithmoderateshaking.
- InoculateavolumeofL-brothcontainedinanappropriatelysizedside-armflaskwithone-tenthvolumeoftheculture(i.e.5mlofcultureto500mlL-broth,2.5mlofculturein250mlL-broth).Growcellsat37°Cwithshaking(200-300rpm)toanOD600of0.5to0.6.
- Chillthecellsinanice-waterbathfor10to15minutesandtransfertoapre-chilled250-mlcentrifugebottle.Dividethecultureifrequired.
- Cellsshouldbekeptat2°Cforallsubsequentsteps.
- Pelletthecellsbycentrifugationat4200rpm,2°Cfor20minutesusingaBeckmanGSArotor.
- PouroffthesupernatantandresUSPendthecellsin.01volumeice-colddH2O(5mldH2Oforanoriginalculturevolumeof500ml;one-halfthisamounttoeachbottleiftheculturewasdividedinstep3.Divideeachsubsequentvolumeinhalfiftheculturewasdivided).Addavolumeofice-colddH2Oequaltotheoriginalculturevolumeandmixwell.Centrifugethecellsasinstep4.
- Pouroffthesupernatantimmediatelyandresuspendthecellsinthesmallamountoffluidremaininginthebottle.
- Thepelletmaybeveryloose.Exercisecareandpouroffthesupernatantimmediately.
- Addanothervolumeofice-colddH2Oequaltotheoriginalculturevolumeandmixwell.Centrifugethecells,againasinstep4.Pouroffthesupernatantimmediatelyandresuspendthecellsintheremainingfluid.
- Ifthecellsaretobeuseddirectlyforelectroporation:
- Placethecellsuspensioninapre-chilled,narrowbottomtubeofappropriatesizeandspinfor10minutesat4200rpm,2°C.
- Decantthesupernatant,andestimatethepellet"svolume.
- Addanequalvolumeofice-colddH2Otoresuspendthecells.
- Keepcellsoniceuntilelectroporationiscompleted.
- Ifthecellsaretobefrozenforlateruse:
- Placethecellsuspensioninanappropriately-sized,narrow-bottomtubethathasbeenpre-chilled.
- Addtothecellsanamountofice-cold10%glycerolequalto0.08oforiginalculturevolume(40mlforacultureoforiginally500ml)andmixwell.
- Spinfor10minutesat4200rpm,2°C.
- Decantthesupernatant,andestimatethepellet"svolume.
- Addanequalvolumeofice-cold10%glycerolandresuspendthecells(onice).
- Aliquot50-300µlofthecellstopre-chilledmicrocentrifugetubes.
- Freezethecellsbyincubationinadryice/ethanolbath.Storeat-80°C.
- Whenreadytouse,thawthecellsonice.
ElectroporationoftheCells
- Settheelectroporationapparatusto2.5kV,25µF.Setthepulsecontrollerto200omega.
- Add1µlplasmidDNAtotubescontaining40µlfreshorthawedcellsonice.MixbyswirlingwithPipettetip.
- ThevolumeofDNAaddedtothecellsshouldbekeptsmall.AddingDNAuptoone-tenththevolumeofcellscanreducetheefficiencyofelectroporation2-to3-fold.
- TransfertheDNAandcellstoapre-chilledelectroporationcuvette(0.2cmelectrodegap)usinganarrowpipettetip.WipeanyiceorwaterfromsidesofcuvetteusingaKimwipe.Placethecuvetteintothesamplechamber.
- Energizetheelectroporationapparatusanddeliverthepulsebypushinginbothchargingbuttonssimultaneouslyandholdinguntilashortbeepisheard.Notethetimeconstantofthepulseandtheactualvoltagedelivered.
- Removethecuvettefromthesamplechamber.Immediatelyadd1mlSOCmediumandtransferthecellstoasterilepolypropyleneculturetubeusingaPasturepipette.
- FailuretoimmediatelyaddSOCtotheelectroporatedcellscansignificantlyreducecellviABIlityanddecreasetransformationefficiency.
- Incubateculturesfor30to60minutesat37°Conarollerorwithmoderateshakingtoallowforplasmidexpression.
- PlatealiquotsoftheelectroporationmixtureonL-agarplatessupplementedwiththeappropriateantibiotics.Incubateplatesat37°C.
SOCBroth(1liter)
Bacto tryptone 20.0 gBacto yeast extract 5.0 gNaCl 0.6 gKCl 0.5 gMgCl2 10 mM (see below)MgSO4 10 mM (see below)Glucose 20 mM (see below)
Note:SOCisidenticaltoSOB,exceptthatitcontains20mMglucose.
- Dissolvetryptone,yeastextract,sodiumchloride,andpotassiumchlorideinafinalvolumeof970mldistilledH2O.Sterilizebyautoclaving.
- Afterautoclaving,allowthesolutiontocoolto60°C,andadd20mlofasterile1Mglucosestock(seebelow)tomakethemedia20mMwithrespecttoglucose.
- Justpriortousing,add10mlofmagnesiumstock(seebelow)totheSOCbrothtomakethemedia20mMwithrespecttomagnesium.
2MMg2+stock(100ml)1MGlucose(100ml)20.3gMgCl218.0gglucose24.7gMgSO4100.0mldH2O
DissolvereagentsinafinalvolumeDissolveglucosein90mldH2O.of100mldH2O.SterilizebyfiltrationBringtofinalvolumeof100ml.througha0.45µmdisposablefilter.SterilizebyfiltrationthroughaTheresultingsolutionis2Mwith0.22µmdisposablefilter.respecttoMg2+.
================ 蚂蚁淘在线 ================
免责声明:本文仅代表作者个人观点,与本网无关。其创作性以及文中陈述文字和内容未经本站证实,对本文以及其中全部或者部分内容、文字的真实性、完整性、及时性本站不做任何保证或承诺,请读者仅作参考,并请自行核实相关内容
版权声明:未经蚂蚁淘在线授权不得转载、摘编或利用其他方式使用上述作品。已经经本网授权使用作品的,应该授权范围内使用,并注明“来源:蚂蚁淘在线”。违反上述声明者,本网将追究其相关法律责任。